Seaweed extract and arbuscular mycorrhiza co-application affect the growth responses and essential oil composition of Foeniculum vulgare L.

The influence of arbuscular mycorrhiza fungi (AMF) inoculation, seaweed extract (SWE) foliar use, and their co-applications were evaluated on the growth-associated traits, antioxidant potential, essential oil profile, and the nutrients content of fennel plants. A factorial experiment was conducted as a completely randomized design with two factors and four replications in the greenhouse. The factors were: AMF inoculation (not inoculated and inoculated with 5 g kg−1) and SWE foliar application (0, 0.5, 1.5, or 3 g L−1). The highest root colonization percentage was recorded in plants treated with AMF + 3 g L−1 of SWE. The top recorded plant height, leaf number, leaf dry weight, biomass, thousand seed weight (TSW), total soluble proteins and total soluble carbohydrates content, antioxidant activity, and essential oil content belonged to AMF + 3 g L−1 of SWE. Furthermore, the co-application of AMF + SWE resulted in a considerable enhancement of the photosynthetic pigments content and, in N, P, K, Fe, Zn, and Mn contents in the shoots and roots. The GC-FID and GC–MS analysis revealed that (E)-anethole (73.28–76.18%), fenchone (5.94–8.26%), limonene (4.64–6.58%), methyl chavicol (2.91–3.18%), and (Z)-β-ocimene (1.36–2.01%) were the principal essential oil constituents. The top (E)-anethole and fenchone contents were obtained by AMF + SWE. Altogether, the simultaneous application of AMF and SWE could be introduced as an environment-friendly strategy to reach reliable growth responses, especially in fennel plants' enriched with some precious essential oil constituents.

www.nature.com/scientificreports/ Photosynthesis pigments content. The results showed that the content of chlorophyll a, b, a + b, and carotenoids content was significantly influenced by SWE and AMF. Foliar application of SWE + AMF inoculation improved photosynthetic pigments content compared to plants sprayed with distilled water and without AMF. The highest of Chl a, b, a + b, and carotenoids content was observed in the plants exposed to SWE foliar application (3 mg L -1 ) + AMF, which increased by 121, 145, 129 and 343% compared to the control plants, respectively (Table 2).
Total soluble proteins content. The findings revealed that the total soluble proteins content was significantly influenced by the simultaneous application of SWE and AMF. The total soluble proteins content increased (up to 60%) in the fennel plant subjected to 3 mg L −1 of SWE + AMF inoculation (Table 3).
Total soluble carbohydrates content. The trait was significantly affected by SWE + AMF employment.
The maximum total soluble carbohydrates content was recorded in the plants supplemented with 3 mg L -1 of SWE + AMF (increased up to 169.5% compared to the control) ( Table 3).
Total antioxidant activity. The total antioxidant activity was significantly increased due to the co-application of SWE + AMF. Under the SWE foliar spraying (3 mg L −1 ) + inoculation of AMF, the total antioxidant activity was enhanced (up to 130.5% compared to the control plants) (Table 3).
Essential oil content. The interactions of SWE + AMF significantly improved the EO content in the fennel seeds. The EO content was enhanced by 114.7% with the co-application of SWE + AMF compared to the control plants (Table 3).

Macro-and micro-nutrients content.
The results showed that macro-and micro-nutrients content was significantly influenced by the foliar application of SWE and AMF inoculation. The N and P content of the shoot and roots as well as K content of the shoots were increased by SWE + AMF. So, the highest recorded data for these traits were recorded at 3 mg L −1 SWE + AMF application (Table 4). While, the lowest N and P content of  www.nature.com/scientificreports/ the shoot and root were observed in the control plants. SWE at 3 mg L −1 with and without AMF attained the highest K content of the root. The co-application of SWE and AMF increased the shoot N, P, and K content by 274%, 331%, and 239%, respectively. Moreover, the co-treatments enhanced the root N, P, and K content by 278, 130, and 275%, respectively. The findings showed that the highest Fe and Zn content of the shoot and root were recorded in the foliar application of SWE (3 mg L −1 ) with and without AMF inoculation and also, in SWE at 1.5 mg L −1 + AMF (Table 5). While, the lowest data for those traits were observed in the control fennel plants, and The influence of arbuscular mycorrhiza fungus (AMF) + seaweed extract (SWE) co-application on plant height, leaf number, leaf dry weight, and thousand seed weight (TSW) of fennel. Dissimilar letters reveal significant differences according to the LSD test at p < 0.05. AMF 0 and AMF 1 assign to those with no mycorrhiza and with mycorrhiza inoculation, and SWE1, SWE2, SWE3, and SWE4 refer to 0, 0.5, 1.5, and 3 mg L −1 of SWE treatment. ) and AMF inoculation. The least content for the mentioned constituent was identified with control plants. The highest limonene content (6.58%) belonged to the foliar SWE treatment (3 g L −1 ) + AMF inoculation. In contrast, the lowest limonene (4.64%) content was recorded for control plants ( Table 6).  Table 4. The influence of seaweed extract (SWE) foliar spraying on macro-nutrients content (mg g −1 DW) of fennel plants under arbuscular mycorrhizal (AMF) inoculation. *, ** indicate significance at P ≤ 5% and ≤ 1%, respectively. Different letters in each column indicate a significant difference at p ≤ 0.05. SWE, S.O.V., and CV assign to seaweed extract, source of variance, and coefficient of variation. AMF 0 and AMF 1 assign to those with no arbuscular mycorrhiza and with arbuscular mycorrhiza inoculation at 5 g kg −1 of soil. www.nature.com/scientificreports/ PCA analysis of the essential oil constituents. The principal component analysis (PCA) of the essential oil composition showed the clear separation of the SWE3 + AMF1 individuals along PC1. The first component explained almost 38% of the total variance, and it was largely loaded by camphene, camphor, pinene, and sabinene (positive correlation) and negatively determined by fenchyl acetate, methyl chavicol, germacrene, limonene, anethole, cymene, and anisaldehyde. In turn, the second component, which explained above 22% of the total variance, facilitated the separation of SWE0 + AMF1 from the other individuals which was characterized by the high content of ocimene, terpinene, thujene, and phellandrene ( Fig. 3).

Discussion
In recent decades, with the development of sustainable agricultural production systems, it's necessary to use nutrient stimulants or symbiotics to enhance plant productivity and to reach a sustainable ecological environment. The stimulants with their specific physiological and molecular actions improve the absorption and efficiency of nutrients and enhance the yield and quality of crops 34 . The application of a variety of these stimulants, especially microbial and non-microbial stimulants, may effectively improve the growth and productivity of plants. In addition, the effects of the combined application of biostimulants may be antagonistic, additive, or synergistic. These effects have encouraged researchers to design and formulate a variety of biostimulants with specific performances concerning their nutritional and functional qualities 35 .
In this study, the simultaneous utilization of AMF and SWE improved plant height, leaf number, leaf dry weight, BY, and TSW of the fennel plants (Table 1). AMF inoculation enhances the growth and biomass of plants, which could be attributed to an increase in internal hormonal levels 36 . It is believed that growth promotion alters the hormonal status of the plant and thus significantly increases nutrient absorption 37 . In agreement with our results, Golubkina et al. 38 showed that using AMF boosted growth traits in the hyssop plant. Likewise, Begum et al. 39 reported that the arbuscular mycorrhiza fungus improved the absorption of water and nutrients in pea plants through a symbiotic relationship and by the creation of an extensive hyphae system. In addition, the improvement of the growth characteristics of the fennel with the application of SWE biostimulant can be assigned to the nutrients and water absorption through the roots and the growth promotion through the action of plant hormones, since, seaweed extract contains micro and macro elements, vitamins, amino acids, and plant hormones such as gibberellin and cytokinin. Also, SWE enhances the division and turgor pressure of meristem cells and hence improves the growth attributes in plants. On the other hand, when sufficient nutrients are available to the plant, the rate of photosynthesis increases, which leads to an enhancement in plant growth and biomass 40,41 . Consistent with our findings, Alam et al. 42 reported that the foliar utilization of SWE had a considerable impact on the growth of roots and shoots of different plant species, including strawberries, winter canola, and coastal pine. Also, Hussain et al. 13 reported that growth traits such as root length and stem and root dry weight of tomato plants were increased by using SWE.
The photosynthetic capacity of leaves directly reflects the level of plant productivity 43 . Total chlorophyll and carotenoid content were also increased by the SWE and AMF treatment. These were compared with the findings of Carrasco-Gill et al., 44 . The use of SWE fertilizer improves the Fe content in the plant tissue, which is one of the essential elements to enhance the chlorophyll content and function in plants 44 . AMF colonization enhances plant photosynthesis potential and thus improves the efficiency of PSII photochemical activity. Similarly, as Table 5. The influence of seaweed extract (SWE) foliar spraying on micro-nutrients content (mg g −1 DW) in fennel plants under arbuscular mycorrhiza (AMF) inoculation. ns, * and ** indicated no significant difference, significant at P ≤ 5% and P ≤ 1%, respectively. Different letters in each column indicate a significant difference at p ≤ 0.05. SWE, S.O.V., and C.V. assign to seaweed extract, source of variance, and coefficient of variation. AMF 0 and AMF 1 assign to those with no arbuscular mycorrhiza and with arbuscular mycorrhiza inoculation at 5 g kg −1 of soil. www.nature.com/scientificreports/ www.nature.com/scientificreports/ formerly described, mycorrhizal plants usually have greater photosynthetic pigments content and chlorophyll fluorescence efficiency, and so attain more tolerance to environmental stresses 45 . Foliar application of the SWE could have increased chlorophyll content via inducing absorption of macro and micronutrients, including Mg and Fe necessary for the synthesis of chlorophyll 46 . Our findings displayed that the co-application of SWE and AMF increased the total protein content in fennel plants. SWE includes several organic ingredients for instance polysaccharides, proteins, and fatty acids which aid in stimulating growth activity (Table 3). Moreover, the protein content of fennel plants was amended with SWE and AMF as a result of increasing nitrate uptake and biological N fixation 47 . SWE and AMF increased total protein content in Trigonella foenum-graecum L. 48 , which agrees with our findings. Also, the improved N uptake and relocation have been repeatedly related to the high protein content 49 . Nonetheless, the increased protein content is related to the enhanced carbohydrate content in plant leaves 50 . The fennel plants treated with SWE and AMF attained a higher carbohydrate content compared to the non-treated ones. The mechanism that has been suggested for AMF concerning plant growth improvement is root mass enhancement that may improve the nutrient absorption and relocation 51 . High total soluble carbohydrates content in the leaves inevitably improves the nitrate assimilation pathway 52 . The enhancement of carbohydrates concentration in AMF-treated plants may be described by the enhanced carbon fixation and the activation of several enzymes 32 . Finally, AMF inoculation improved the net photosynthetic potential as well as the carbohydrate content in Zenia plants 53 .

SWE (mg L −1 ) AMF
In the present experiment, it is realized that the essential oil content significantly increased due to the coapplication of AMF and SWE. AMF improves metabolism and affects the content and quality of secondary metabolites 54 . An indirect mechanism can be influential through the effect on the microbial community and the absorption of the elements in the soil on the content and yield of the EO 55 . On the other hand, the enhanced EO biosynthesis can also be justified by the absorption of the P element, which happened as a result of AMF inoculation 56 . The increase in EO content can also be due to the presence of growth stimulants in the SWE treatment. The nutrients in SWE, such as boron and nitrogen, stimulate the essential oil accumulation in plants 57 . Other nutrients such as phosphorus, and micro-elements like Zn, may induce the growth and enhance the essential oil composition of plants supplemented with SWE 18 . In agreement with our findings, SWE enhanced the EO content and constituent in hyssop plants 58 . Also, Tawfeeq et al. 59 showed that SWE treatment increased EO compounds (a-terpinene and a-phellandrene) in rosemary. Similarly, the highest content of EO in Dutch fennel was traced as a result of the co-application of arbuscular mycorrhizal fungus and seaweed extract 60 .
In the current research, N, P, K, Fe, Zn, and Mn contents were improved in the fennel plants supplemented with AMF + SWE. The reason for the increased micronutrients content can be ascribed to the high absorption potential as a result of the symbiosis with the mycorrhizal fungus in addition to the high plant root surface area which also improves the nutrients absorption efficacy. By creating a symbiotic relationship between plants and fungi, AMF improves the uptake of essential elements such as phosphorus and nitrogen by fungal hyphae and simultaneously enhances the nutrients contained in the plant 61 . SWE is also rich in nutrients, such that it has a variety of macro and micro-elements 62 . As a result, the significant enhancement of N, P, K, Fe, Zn, and Mn elements concentration can also be attributed to the additional availability of nutrients. In addition, SWE comprises plant hormones that induce the growth of roots and greatly boost nutrient uptake 63 . Our results are in agreement with the findings of Baslam, et al. 64 and Abbas et al. 65 . Whether the observed growth stimulation is attributed to the macro-or micro-nutrition available in SWE or to the growth stimulants (i.e. plant hormones) remains to be seen. www.nature.com/scientificreports/

Materials and Methods
Study site and treatment. The research was run in the greenhouse of Maragheh University, Iran, which is located at 37°23′ north latitude and 46°16′ east longitude, and 1486 m above sea level. The temperature regime was 18-25°C for the night-day, relative humidity was nearby 60-70% and air-conditioned with fans with a 0.5-1.5 m s -1 . A factorial experiment in the format of a completely randomized design (CRD), with four replications, and three plants per replication was set up. The current research had two factors including the fungus F. mosseae inoculation (0 and 5 g kg −1 of soil) to the soil at the planting moment and, SWE foliar spraying was applied at 4 levels (0, 0.5, 1.5, and 3 g L −1 ). The AMF was acquired from Zist Fanavar Pishtaz Varian, Karaj, Iran, with one hundred active spores per gram of soil. The SWE (MAX Sailor) was provided by CITYMAX company, Xian, China, which contained organic acid (50%), alginic acid (16%), P (16%), amino acid (16%), N (1%), mannitol (3%), gibberellic acid (300 ppm), auxin (0-20 ppm) and cytokinin (0-30 ppm), and pH was 9-11. The foliar use of SWE was accomplished 30 days after sowing and repeated three times at 7-day intervals. The control fennel plants were grown in the conditions without AMF inoculation and SWE foliar application and they were sprayed with distilled water instead of SWE. The fennel plants were foliar sprayed till the solution or distilled water drops were run off the shoots. 10-20 ml of the SWE treatments on average was foliar sprayed on each plant based on the fennel growth stage. The utilized soil was autoclaved at 121°C under a 1.2 atmosphere for 90 min, to eradicate its microorganisms before sowing the fennel seeds (F. vulgare Miller). The fennel seeds were planted in 5 L pots. The fennel plants were watered with tap water every 3-4 days. The soil was sandy clay loam with pH = 8.07, 0.96% organic carbon, 0.08% total N, and 9.06, 485.62, 1.12, 192, and 1.06 mg kg −1 of available P, K, Zn, Mn, and Fe, respectively. All methods in the current study were performed by the relevant institutional, national, and international guidelines and legislation. Total soluble proteins content. 0.2 g of fresh leaf samples were powdered via liquid nitrogen and squelched in 1.5 ml of sodium phosphate buffer (100 mM and pH = 7.8) along with 1 mM EDTA and 2% (w/v) polyvinyl pyrrolidone. Then, it was centrifuged for 15 min at 4°C and 12,000 rpm, and the supernatant fluid was analyzed to estimate the total soluble proteins content based on the method of Bradford 68 . Finally, the OD was measured at 595 nm with a spectrophotometer and presented as mg g -1 FW.

Microscopic imaging of root colonization.
Total soluble carbohydrates content. 0.2 g of fresh fennel leaves sample were crushed using 10 ml of ethanol (95%) for about 1 h in a water bath at a temperature of 80℃ and centrifuged at 12,000 rpm for 10 min. After that, a mixture was made with 1 ml of the supernatant, phenol (0.5%), and 5 ml of sulfuric acid (98%). Finally, the OD was noted at 483 nm via a spectrophotometer. The total soluble carbohydrates content was reported as mg g −1 FW 69 . www.nature.com/scientificreports/ gramming was: 5 min at 60°C, reaching 240°C at 3°C/min ramp, held for 10 min at the temperature. The helium (carrier gas) flow rate was 1 mL/min; the injector split ratio was 1:30; and the mass range and electron impact (EI) were 40-400 m/z and 70 eV, respectively. The oil constituents were identified by Adams 72 according to an interactive combination of linear retention indices (RIs), calculated against a homologous series of n-alkanes (C8-C40, Supelco, Bellefonte, CA, USA) and mass spectrum (MS) matching with libraries (ADAMS, WILEY 275 and NIST 17). The GC-FID analysis was carried out by an Agilent 7990 B gas chromatography connected to a flame ionization detector (FID), capillary column VF 5MS (30 m, 0.25 mm i.d., 0.50 μm f.t., 5% phenyl methylpolysiloxane). The above-mentioned oven temperature programming was employed. The injection volume was 1μl of 1:100 (oil: hexane). The quantification of the oil components was carried out by considering the peak area normalization without correction factors 72 .

Measurement of the total antioxidant activity.
Macro-and micro-elements content estimation. A flame photometer was employed to evaluate K content in samples. Also, N and P content were measured through Kjeldahl and the yellow technique, respectively. Vanadate molybdate was applied as an indicator of the yellow method. The amount of P was assessed at a wavelength of 470 nm by a spectrophotometer 73 . Mn, Fe, and Zn contents were determined using an atomic absorption spectrometer (AA-6300 F; Shimadzu, Kyoto, Japan) 74 .

Statistical analysis.
The MSTAT-C ver. 2.1 was employed for the analysis of variance, and the least significant difference test (LSD) at 1% and 5% probability levels were applied for the comparisons of the evaluated traits means. Statistica ver. 13.3. software (TIBCO Software Inc. 2017, Palo Alto, CA, USA) was used to perform principal component analysis (PCA) of the essential oil constituents.
Ethics approval and consent to participate. All procedures were conducted according the relevant institutional, national, and international guidelines and legislation.

Conclusions
Overall, the simultaneous application of SWE and AMF enhanced the growth characteristics, protein, carbohydrates, macro-and micro-nutrients contents, and EO composition of the fennel plants. Therefore, the co-treatment of the SWE foliar application + AMF symbiosis can be advised as a promising tool to improve the fennel growth and EO composition. Furthermore, the co-application of AMF and SWE may be considered a reliable biofertilization methodology. All in all, the idea is that the co-application of these biofertilizers, not only reduces the chemical fertilizers' input and their related adverse impression on the ecosystem but also improves the qualitative and quantitative attributes of fennel plants.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.